While trichloroethylene (TCE) has widely been used as solvent, it is toxic and difficult to decompose by natural microbes, whereby problems of the pollution of the soil and subterranean water therefrom have been brought about in various districts.
A technique has been proposed in WO89/09827 for decomposing TCE, in which a toluene-monoxygenase gene originating from Pseudomonas mendocina KR-1 and having a biodegradability for TCE is introduced into a host bacterial cell by a foreign plasmid and the bacteria having this recombinant gene is used to effect biodegradation of TCE. However, this recombinant is obtained by introduction of a toluene-monoxygenase gene by a foreign plasmid and the toluene-monoxygenase gene is not carried on the chromosome, so that the efficiency of the biodegradation of trichloroethylene is low.
A technique of the biodegradation of TCE using a recombinant by recourse also to the use of a foreign plasmid is reported in the Journal of Fermentation and Bio-engineering, Vol. 79, No. 2, 100-106 (1995). This technique is based on the use of the phenol-hydroxylase gene of Pseudomonas putida BH. However, this recombinant also exhibits a low capacity for decomposing chlorinated ethylene per unit cell population of as low as about 7.5 mg/liter.multidot.A.sub.600 (A.sub.600 is an absorbancy at 600 nm and serves as a parameter for the bacterial cell concentration). It is taught therein that the phenol-hydroxylase gene of Pseudomonas putida BH is composed of several components. It is also indicated in J. Ferment. and Bioeng., Vol. 79, No. 5, 485-488 (1995) that the phenol-hydroxylase gene of Pseudomonas putida BH is composed of several components.
In Japanese Patent Kokai Hei-6-105691 A, it is described that the phenol-hydroxylase (hereinafter sometimes abbreviated as PH) which is originated from the chromosomal DNA of the cell strain Pseudomonas putida KWI-9 decomposes trichloroethylene. Herein is described that a recombinant is prepared by introducing into the host cell a recombinant DNA having the PH gene and, downstream thereof, a terminator connected thereto, by a foreign plasmid. However, the capability of this recombinant for decomposing chlorinated ethylene is not sufficient, since the recombinant DNA is carried on the foreign plasmid.
Afterwards, it was confirmed by research at the gene level for the PH that the minimum unit of the PH gene for trichloroethylene-biodegradability consists of a group of chlorinated ethylene decomposing genes composed of 5 genes represented as pheA, pheB, pheC, pheD and pheE and that a gene for facilitating decomposition of chlorinated ethylene, which is denoted as pheZ, is present upstream from them. This bacteria Pseudomonas putida KWI-9 has the PH gene on the chromosomal DNA and exhibits a capability for biodegradation of chlorinated ethylene, though at a low level.
In Report of Environmental Engineering, Vol. 33, 165-175 (1996), a technique is given in which a tac-promotor is inserted upstream of the PH gene on the chromosome of the cell strain Pseudomonas putida KWI-9 to formulate a recombinant (Pseudomonas putida KN1-10A) which is used for biodegradation of trichloroethylene. However, this recombinant exhibits a low capacity for the biodegradation of chlorinated ethylene per unit cell population, since a promoter is inserted within the gene for facilitating decomposition of chlorinated ethylene, i.e. pheZ, and thus, the function of pheZ is obstructed.
Thus, recombinants of the prior art exhibit lower capabilities for decomposing chlorinated ethylene due to blocking of the function of the gene pheZ which facilitates decomposition of chlorinated ethylene or because of a biodegradability based on the function of a foreign plasmid, so that there has been a request for an improvement in the biodegradability for chlorinated ethylene.